Casein Coated Drug-Loaded Iron Oxide Nanoparticles

ABSTRACT

This disclosure relates to nanoparticle drug delivery systems composed of casein (CN) coated nanoparticles, e.g., iron oxide nanoparticles coated with an inner layer and an out layer comprising the milk protein casein. In certain embodiments, drug molecules are incorporated into an inner polymeric layer coating the nanoparticles, which are subsequently coated with a casein containing outer layer, i.e., a layer-by-layer (LBL) construction. Oral administration of these casein coated nanoparticles are contemplated as experiments indicated sufficiently stability in conditions that simulate the conditions of the gut. Drugs that were loaded into the nanoparticle systems were released when the casein outer layer was gradually degraded in the presence of an intestinal protease meant to simulate conditions of the intestine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/882,482filed Oct. 14, 2015, which claims the benefit of U.S. ProvisionalApplication No. 62/064,231 filed Oct. 15, 2014. The entirety of each ofthese applications is hereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under R01CA154846-02 andU01CA151810-02 grants awarded by the NIH. The government has certainrights in the invention.

BACKGROUND

Feridex® contains iron oxide particles associated with dextran for usedas a magnetic resonance imaging contrast media. Feraheme™ (ferumoxytol)is an anemia drug that contains iron oxide particles surrounded by apolyglucose sorbitol carboxymethylether coating. Both Feridex® andFeraheme™ are intravenous formulations.

Oral delivery is considered an ideal drug administration route becauseit not only avoids the discomfort and additional procedures associatedwith intravenous delivery injections but also allows for delivery ofnon-water-soluble drugs. However, oral delivery through organs in thegastrointestinal (GI) tract needs to overcome several obstacles,including: 1) the strong acidic gastric environment that reduces thedrug stability and solubility; 2) the digestive enzymes that degradedrugs and decrease drug bioavailability; and 3) a mucus barrier thatblocks drug penetration and subsequent tissue absorption. Even if a drugcan be formulated for oral administration, it remains a challenge todeliver the drugs to a specific segment of the GI tract, such as theintestine, for maximal drug action. In clinical practice, certain drugs,for example, those for treating Crohn's disease, ulcerative colitis andchemotherapy medications, may need controlled release of the drug in thetargeted areas or organs of the GI tract to increase the bioavailabilityand efficacy of the drug while reducing the toxicity to the normalorgans and tissue. Thus there is a need to identify improved oralformulations.

Casein (CN) is a major protein ingredient in milk and can formmicelle-like porous structures with the capacity of absorbing vitaminsand minerals, for nutrient delivery. Huang et al. report casein-coatediron oxide nanoparticles for high MRI contrast enhancement and efficientcell targeting. ACS Appl Mater Interfaces, 5 (2013), pp. 4632-4639.

Ying et al. report the evaluation of the cytotoxicity of iron oxidenanoparticles with different coatings and different sizes. Science ofthe Total Environment, 408 (2010) 4475-4481.

Zuo et al. report PEM-coated alginate microgels for controlled releaseof protein. Biomed Mater, 7 (2012)

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to nanoparticle drug delivery systems composedof casein (CN) coated nanoparticles, e.g., iron oxide nanoparticlescoated with an inner layer and an out layer comprising the milk proteincasein. In certain embodiments, drug molecules are incorporated into aninner polymeric layer coating the nanoparticles, which are subsequentlycoated with a casein containing outer layer, i.e., a layer-by-layer(LBL) construction. Oral administration of these casein coatednanoparticles are contemplated as experiments indicated sufficientlystability in conditions that simulate the conditions of the gut. Drugsthat were loaded into the nanoparticle systems were released when thecasein outer layer was gradually degraded in the presence of anintestinal protease meant to simulate conditions of the intestine.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising a particle; an inner coating on the particlecomprising an amphiphilic polymer providing a hydrophobic space forembedding a hydrophobic drug within the inner coating, wherein ahydrophobic drug is in the space of the inner coating; and an outercoating over inner coating wherein the outer coating is crosslink caseinmolecules that are hydrophilic, making the whole nanoparticles watersoluble.

In certain embodiments, the disclosure relates to methods of treating adisease or condition comprising administering an effective amount of apharmaceutical composition of disclosed herein to a subject in needthereof. In certain embodiments, the disease is cancer and thehydrophobic drug is an anticancer drug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme that illustrates layer-by-layer assembly of caseincoated iron oxide nanoparticles loaded with a drug(Doxorubicin/Indocyanine green).

FIG. 2A shows data on DLS profiles of size distribution of SHP-IO,DOX-IO, CN-DOX-IO.

FIG. 2B shows gel electrophoresis of SHP-IO, DOX-IO, CN-DOX-IO (upper),and corresponding GelCode Blue staining for the presence of caseincoating.

FIG. 2C shows data on UV-Vis absorption spectra of SHP-IO, DOX-IO andCN-DOX-IO with distinctive peak of DOX indicated.

FIG. 3A shows data on the changes of the hydrodynamic size at differentpH of CN-DOX-IO nanoparticles measured by DLS.

FIG. 3B shows data on the changes of the hydrodynamic size at differentpH of DOX-IO nanoparticles measured by DLS.

FIG. 3C shows SDS-PAGE analysis of digested protein fragments from thecoating layer of CN-DOX-IO after treated with gastric enzyme pepsin atpH 2.0

FIG. 3D shows SDS-PAGE analysis of digested protein fragments from thecoating layer of CN-DOX-IO after treated with small intestine enzymetrypsin at pH 7.

FIG. 3E shows data on changes of hydrodynamic sizes of CN-DOX-IO aftertreated with pepsin at pH 2.0 at different enzyme concentrations

FIG. 3F shows data on changes of hydrodynamic sizes of CN-DOX-IO aftertreated with trypsin at pH 7.0 at different enzyme concentrations.

FIG. 4A shows data on release profiles of DOX from CN-DOX-IO and DOX-IOat different pH, in the simulated gastric juice (pH 2.0, [Pep]=1.0mg/mL).

FIG. 4B shows data on release profiles of DOX from CN-DOX-IO and DOX-IOat different pH, in the simulated intestinal juice (pH 7.0, [Try]=2.5mg/mL).

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

As used herein, “casein” refers to a group of casein proteins (αs1, β,αs2 and κ) found in milk as the major components, which are coded by thegenes (CSN1S1, CSN2, CSN1S2 and CSN3, respectively). The dominantfeature of milk is the casein micelle, a supramolecular aggregateimparts the white characteristic of milk. Because αs1,2-caseins andβ-caseins are highly phosphorylated, they are believed to bind withcalcium to form the aggregates. κ-casein is thought to predominate onthe micellar surface. Casein may be purified from milk. Casein exists inmilk as the calcium salt, calcium caseinate. Calcium caseinate has itsisoelectric point at pH lower than the pH of milk; therefore the caseinmicelle is solubilized. If acid is added to milk, the caseinprecipitates. Further extraction with ethanol allows for furtherpurification. Experiments performed herein utilize casein from bovinemilk. In certain embodiments, the disclosure contemplates that casein isderived from other animals such as humans, buffaloes, goats, camels andsheep. In certain embodiments, the disclosure contemplates that thecasein proteins may be produced by recombinant methods.

In certain embodiments, the disclosure contemplates pharmaceuticalcompositions comprising casein containing particles disclosed herein anda pharmaceutically acceptable excipient typically in the form of a pill,hard or soft shell capsule, tablet, gel, oral powder, or liquidformulation. Liquid and solid preparations for oral use may containsuitable antimicrobial preservatives, antioxidants and other excipientssuch as dispersing, suspending, thickening, emulsifying, buffering,wetting, solubilizing, stabilizing, flavoring and sweetening agents.Liquid vehicle may include sucrose or a suitable polyhydric alcohol oralcohols and which optionally contain ethanol, an elixir or linctus.

Examples of excipients include polysaccharides, petrolatum, gelatin, andmineral oil, antiadherents, binders, coatings, colors, disintegrants,flavors, glidants, lubricants, preservatives, sorbents, and sweeteners.Typical binders include saccharides, disaccharides: sucrose, lactose;polysaccharides starches, cellulose or modified cellulose such asmicrocrystalline cellulose and cellulose ethers such as hydroxypropylcellulose (HPC); xylitol, sorbitol or maltitol; gelatin;polyvinylpyrrolidone (PVP), polyethylene glycol (PEG). Solution bindersinclude gelatin, cellulose, cellulose derivatives, polyvinylpyrrolidone,starch, sucrose and polyethylene glycol. Dry binders include cellulose,methyl cellulose, polyvinylpyrrolidone and polyethylene glycol. Typicalsweeteners include a saccharide like citric acid and sodium citrate.Typical preservatives include antioxidants like vitamin A, vitamin E,vitamin C, retinyl palmitate, and selenium, or amino acids like cysteineand methionine and methyl paraben and propyl paraben. Typical lubricantsinclude talc or silica, and fats, e.g. vegetable stearin, magnesiumstearate or stearic acid. Typical glidants include fumed silica, talc,and magnesium carbonate. Typical disintegrants include crosslinkedpolyvinylpyrrolidone (crospovidone), crosslinked sodium carboxymethylcellulose (croscarmellose sodium) and sodium starch glycolate. Typicalcoatings include a cellulose ether hydroxypropyl methylcellulose (HPMC),shellac, gelatin, or polysaccharides.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” may be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample compound compared to a control without the compound. It may alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers.

The cancer to be treated in the context of the present disclosure may beany type of cancer or tumor. These tumors or cancer include, and are notlimited to, tumors of the hematopoietic and lymphoid tissues orhematopoietic and lymphoid malignancies, tumors that affect the blood,bone marrow, lymph, and lymphatic system. Hematological malignancies mayderive from either of the two major blood cell lineages: myeloid andlymphoid cell lines. The myeloid cell line normally producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells;the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas,lymphocytic leukemias, and myeloma are from the lymphoid line, whileacute and chronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin.

Also contemplated are malignancies located in the colon, abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvis, skin, soft tissue, spleen, thorax and genito-urinaryapparatus and, more particularly, childhood acute lymphoblasticleukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia,acute myeloid leukemia, adrenocortical carcinoma, adult (primary)hepatocellular cancer, adult (primary) liver cancer, adult acutelymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin'sdisease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adultnon-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissuesarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, analcancer, astrocytoma, cancer of the biliary tract, cancer of the bladder,bone cancer, brain stem glioma, brain tumors, breast cancer, cancer ofthe renal pelvis and ureter, primary central nervous system lymphoma,central nervous system lymphoma, cerebellar astrocytoma, brainastrocytoma, cancer of the cervix, childhood (primary) hepatocellularcancer, childhood (primary) liver cancer, childhood acute lymphoblasticleukemia, childhood acute myeloid leukemia, childhood brain stem glioma,childhood cerebellar astrocytoma, childhood brain astrocytoma, childhoodextracranial germ cell tumors, childhood Hodgkin's disease, childhoodHodgkin's lymphoma, childhood visual pathway and hypothalamic glioma,childhood lymphoblastic leukemia, childhood medulloblastoma, childhoodnon-Hodgkin's lymphoma, childhood supratentorial primitiveneuroectodermal and pineal tumors, childhood primary liver cancer,childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhoodvisual pathway and hypothalamic glioma, chronic lymphocytic leukemia,chronic myeloid leukemia, cancer of the colon, cutaneous T-celllymphoma, endocrine pancreatic islet cells carcinoma, endometrialcancer, ependymoma, epithelial cancer, cancer of the oesophagus, Ewing'ssarcoma and related tumors, cancer of the exocrine pancreas,extracranial germ cell tumor, extragonadal germ cell tumor, extrahepaticbiliary tract cancer, cancer of the eye, breast cancer in women,Gaucher's disease, cancer of the gallbladder, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal tumors, germ celltumors, gestational trophoblastic tumor, tricoleukemia, head and neckcancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma,hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers,intraocular melanoma, islet cell carcinoma, islet cell pancreaticcancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancerof the lip and mouth, cancer of the liver, cancer of the lung,lymphoproliferative disorders, macroglobulinemia, breast cancer in men,malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma,mesothelioma, occult primary metastatic squamous neck cancer, primarymetastatic squamous neck cancer, metastatic squamous neck cancer,multiple myeloma, multiple myeloma/plasmatic cell neoplasia,myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia,myeloproliferative disorders, paranasal sinus and nasal cavity cancer,nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma duringpregnancy, non-melanoma skin cancer, non-small cell lung cancer,metastatic squamous neck cancer with occult primary, buccopharyngealcancer, malignant fibrous histiocytoma, malignant fibrousosteosarcoma/histiocytoma of the bone, epithelial ovarian cancer,ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancerof the penis, phaeochromocytoma, hypophysis tumor, neoplasia ofplasmatic cells/multiple myeloma, primary central nervous systemlymphoma, primary liver cancer, prostate cancer, rectal cancer, renalcell cancer, cancer of the renal pelvis and ureter, retinoblastoma,rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas,skin cancer, small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous neck cancer, stomach cancer, pineal and supratentorialprimitive neuroectodermal tumors, T-cell lymphoma, testicular cancer,thymoma, thyroid cancer, transitional cell cancer of the renal pelvisand ureter, transitional renal pelvis and ureter cancer, trophoblastictumors, cell cancer of the renal pelvis and ureter, cancer of theurethra, cancer of the uterus, uterine sarcoma, vaginal cancer, opticpathway and hypothalamic glioma, cancer of the vulva, Waldenstrom'smacroglobulinemia, Wilms' tumor and any other hyperproliferativedisease, as well as neoplasia, located in the system of a previouslymentioned organ.

A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent” or thelike, refer to molecules that are recognized to aid in the treatment ofa cancer. Contemplated examples include the following molecules orderivatives such as temozolomide, carmustine, bevacizumab, procarbazine,lomustine, vincristine, gefitinib, erlotinib, cisplatin, carboplatin,oxaliplatin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed,methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin, mithramycin, vinblastine, vindesine, vinorelbine,paclitaxel, taxol, docetaxel, etoposide, teniposide, amsacrine,topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene,raloxifene, droloxifene, iodoxyfene, fulvestrant, bicalutamide,flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin,megestrol, anastrozole, letrozole, vorozole, exemestane, finasteride,marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin,thalidomide, azacitidine, azathioprine, capecitabine, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, doxifluridine, epothilone,irinotecan, mechlorethamine, mercaptopurine, mitoxantrone, pemetrexed,tioguanine, valrubicin and/or lenalidomide or combinations thereof suchas cyclophosphamide, methotrexate, 5-fluorouracil (CMF); doxorubicin,cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone(MOPP); sdriamycin, bleomycin, vinblastine, dacarbazine (ABVD);cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP);bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin,5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX);methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “subject” refers to any animal, preferably a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

Casein Protein Coated Magnetic Nanoparticle Enabled Oral Drug Delivery

This disclosure relates to nanoparticle drug delivery systems composedof casein (CN) coated nanoparticles, e.g., iron oxide nanoparticlescoated with an inner layer and an out layer comprising the milk proteincasein. In certain embodiments, drug molecules are incorporated into aninner polymeric layer coating the nanoparticles, which are subsequentlycoated with a casein containing outer layer, i.e., a layer-by-layer(LBL) construction. Oral administration of these casein coatednanoparticles are contemplated as experiments indicated sufficientlystability in conditions that simulate the conditions of the gut. Drugsthat were loaded into the nanoparticle systems were released when thecasein outer layer was gradually degraded in the presence of anintestinal protease meant to simulate conditions of the intestine.

A pH stable and enzymatic-responsive oral drug delivery nanoparticlesystem with MRI visible contrast has been developed via a layer-by-layerdesign, using modified milk protein casein to form an outer layer thatprotected the hydrophobic drug loaded in the inner polymer coating layerwhich caps the magnetic iron oxide nanoparticle core. The casein outerlayer is resistant to degradation by protease pepsin at low pH undergastric conditions, and can be disassembled by the small intestineenzyme trypsin at neutral pH. Therefore, small intestine targeted drugdelivery can be achieved by reducing the pre-mature drug release in theacidic stomach and then conducting the enzymatic-responsive release inthe small intestine. Furthermore, this nanoconstruct retains the abilityto provide an effective MRI contrast enhancing effect, providing thepotential capability of MRI monitored and/or magnetic directed drugdelivery. Given high water solubility, pH stability and enzymaticresponsiveness as well as excellent biocompatibility, the reported LBLCN-DOX-IO is a promising drug delivery system for oral delivery ofhydrophobic drugs, capable of by-passing low stomach pH and enablingabsorption in the lower GI tract with neutral pH.

The structured nanocarriers presented herein provide an approach forpreferential drug delivery to the intestine, with good stability in thelow pH stomach fluid and enhanced mucosal/membrane penetration, mainlyattributed to the casein coating of the nanostructure. Most layeredstructures are microsized or have positive surface charge, which areunstable in the acidic stomach, thus not suitable for theintestinal-specific drug delivery. Experiments herein indicate that, theLBL CN-DOX-IO nanoparticles show a significant reduction of the initialrapid release of DOX from the amphiphilic inner polymer layer in the lowpH conditions of the stomach fluid. Therefore, a higher amount ofpayload drug was retained for delivery and release in the intestine.

In addition to the protective function of the outer casein layer, caseinenhances the cellular uptake. Significant enhanced permeability of LBLCN-DOX-IO was observed in the ex vivo experiment using small intestinesacs treated with different nanoconstructs, in which we observed moreLBL CN-DOX-IO delivered deeply into the villi pits compared to DOX-IOwithout the casein outer layer. Although the mechanism by which caseinimproved the cell uptake and tissue penetration remained unclear and itis not intended the embodiments of this disclosure be limited by anyparticular mechanism, the LBL CN-DOX-IO likely has the ability topenetrate the mucus, which is one of the major obstacles for intestinaldrug delivery. This enhanced absorption in intestinal villi was furtherconfirmed by the histological analysis with Prussian blue staining. Bluedots stained by iron could be observed in the intestinal villi 3 h afteroral administration of the LBL construct, but not in those treated withDOX-IO.

In vivo monitoring of drug delivery with non-invasive imaging has becomea desirable tool for planning and evaluating the therapeutic strategy aswell as optimizing individualized treatments. Most development andinvestigation of the intestinal drug delivery systems are dependent onthe in vitro assessment/evaluation with conventional methods, such ascellular uptake and mucus penetration. Drug delivery systems combinedwith imaging probes for imaging strategies, such as QDs, radioactivemoieties and NIR dyes, have recently been developed for image-guidedassessment. Magnetic nanoparticles act as a core for the LBL structure,which provides a template for the LBL assembly and offers MRI contrastenhancement. As a result, the LBL nanoconstruct is useful for MRImonitoring drug delivery. Furthermore, magnetic iron oxide nanoparticlespresent potential capabilities of thermal-induced drug release andmagnetic localization to improve the drug delivery with the reported LBLdrug delivery system.

In certain embodiments, magnetic iron oxide nanoparticle cores can beused as an MRI contrast agent for in vivo imaging and guided drugdelivery. Thus in certain embodiments, the disclosure contemplatesmethods of imaging comprising administering drug delivery systemsdisclosed herein to a subject in need thereof and exposing an area ofthe subject, e.g. an area of suspected cancer growth such as a tissue,organ, or the circulatory system, to an induced magnetic field underconditions such that magnetic resonance can detect the core of the ironoxide nanoparticle at a location and generating an image that identifiesthe location of the core in relation to other surrounding cells,tissues, organs, liquids, or bones.

In certain embodiments, the disclosure contemplates generatingsufficiently localized heat or hypothermia in an area or location of theimaged coated iron oxide nanoparticles to cause cell wall or tissuebreakdown or destruction.

The utilization of DOX and ICG were to model drug molecules. Hydrophobicdrugs to specific diseases can be applied with the reported LBLconstruct.

In certain embodiments, the nanoparticle systems may be targeted tocells, tissues, organs, or bones by covalent attachment of a specificbinding agent, e.g., that binds a cell surface marker, to the outerlayer.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising a particle; an inner coating on the particlecomprising an amphiphilic polymer providing an space for embedding ahydrophobic drug within the inner coating, wherein a hydrophobic drug isin the space of the inner coating; and an outer coating on the innercoating wherein the outer coating is crosslink casein molecules.

In certain embodiments, the particle is an iron oxide nanoparticle. Incertain embodiments, the amphiphilic polymer comprises maleic acid andoctadecene monomers.

In certain embodiments, the hydrophobic drug is an anticancer drug. Incertain embodiments, the anticancer drug is doxorubicin. In certainembodiments, the crosslinked casein molecules are made by the process ofmixing casein and the particle comprising the inner coating in thepresences of glutaraldehyde.

In certain embodiments, the disclosure relates to methods of treating adisease or condition comprising administering an effective amount of apharmaceutical composition of disclosed herein to a subject in needthereof. In certain embodiments, the disease is cancer and thehydrophobic drug is an anticancer drug.

Examples Magnetic Iron Oxide Nanoparticles (SHP-IO)

Iron oxide nanoparticles with a monolayer of oleic acid and an averagecore diameter of 10 nm are mixed with a hydrolyzed copolymer ofpoly(maleic acid) and octadecene to provide an amphiphilic coating. SeeDuan et al. J. Phys. Chem. C, 2008, 112 (22), pp 8127-8131.

Preparation of LBL Casein Coated IO Nanoparticles Loaded withDoxorubicin

The process for preparing the layer-by-layer (LBL) assembled caseincoated iron oxide nanoparticles loaded with drugs (DOX/ICG)(CN-DOX/ICG-IO) is illustrated in the scheme of FIG. 1. The inner layerof amphiphilic polymer offered a coating layer for the 10 nanoparticlecore along with the space for embedding the hydrophobic small moleculesDOX/ICG. CN was then deposited and assembled into an outer layer. SHP-IOnanoparticle suspension (1 mg/mL) was mixed with freshly prepareddoxorubicin solution in methanol (1 mg/mL). The mixture was shaken andincubated for 2 h so that hydrophobic DOX could be incorporated into thehydrophobic layer of the amphiphilic coating polymer. Doxorubicin-loadediron oxide nanoparticles (DOX-IO) were collected by centrifuging using acentrifuge tube with a cut-off size of 100 kDa. Collected DOX-IO wasrinsed several times with deionized water until no free DOX was detectedin the rinsing solution.

Formation of LBL CN-DOX-IO Nanoparticles

DOX-IO solution was mixed with casein (CN) solution at the weight ratioof Fe:CN=1:2. The mixture was kept at room temperature for 24 h to allowcasein molecules to assemble on the surface of DOX-IO. Then a freshlyprepared 0.4% glutaraldehyde solution was added to crosslink the caseinmolecules to form an outer layer on the surface of DOX-IO. After 2 h,the product of casein coated doxorubicin-loaded iron oxide nanoparticles(CN-DOX-IO) was collected by centrifuging using a centrifuge tube with acut-off size of 100 kDa, and washed with deionized water three times.

The core diameters, hydrodynamic sizes and zeta potentials of theprepared DOX-IO and CN-DOX-IO were determined by transmission electronmicroscope (TEM, HitachiH-7500, accelerating voltage 75 kV) and dynamiclight scattering (DLS, Malvern Zeta Sizer Nano S-90) instrument,respectively. Gel electrophoresis was performed to confirm the presenceof casein coating in CN-DOX-IO using 2% agarose gel. The percentage ofDOX loaded on IONPs was determined by the weight ratio of loaded DOX toFe. Samples were dissolved in 1 m HCl, and sonicated for 30 min, andthen measured for the fluorescence intensity from DOX at 590 nm (λex=485nm) with a microplate reader (Synergy 2 Multi-Mode Microplate Reader,BioTek, USA) to determine the amount of loaded DOX. The Fe concentrationwas determined by the phenanthroline-colorimetric method.

From TEM images of SHP-IO, DOX-IO and LBL CN-DOX-IO nanoparticles(FIG. 1) the average diameters of the IO nanoparticle cores, measuredfrom 100 individual nanoparticles in the TEM images, are calculated tobe: 10.1±0.6, 10.4±0.5, and 10.4±0.6 for SHP-IO, DOX-IO and CN-DOX-IO,respectively. This indicates the core size of IO nanoparticles remainedunchanged after DOX loading in the inner polymeric layer and subsequentassembly of the casein outer layer. The formation of LBL CN-DOX-IO wasconfirmed by the increased hydrodynamic sizes with increasing coatinglayers. After coating with amphiphilic polymer, hydrophobic IOnanoparticles were transferred into aqueous solution, and consequentlythe hydrodynamic size increased from 10.1 nm (TO, data not shown) to18.8 nm (SHP-IO). However, the hydrodynamic size of DOX-IO (17.7±3.8 nm)was slightly smaller compared with that of SHP-IO, which was attributedto the contraction of the polymer coating when DOX was absorbed in themethanol/water mixture. The hydrodynamic sizes increased to 24.4±4.67 nm(CN-DOX-IO) after applying the outer layer of CN (FIG. 2A). Furthermore,gel electrophoresis demonstrated a higher molecular weight and lowermobility of LBL CN-DOX-IO comparing to that of DOX-IO and SHP-IO (FIG.2B). It is notable that DOX-IO showed the highest mobility inelectrophoresis because of its smallest size. Subsequently staining thegel with GelCode Blue evidenced a marked blue band in CN-DOX-IO,indicating the presence of protein coating on CN-DOX-IO. In contrast, nosuch band was observed for SHP-IO and DOX-IO (FIG. 2B). The UV-Visabsorption spectra of both DOX-IO and CN-DOX-IO revealed thecharacteristic peak of DOX at 495 nm, as shown in FIG. 2C, representingthat DOX was successfully loaded on DOX-IO and remained in the LBLCN-DOX-IO structure when the casein outer layer formed. All three typesof nanoparticles exhibited excellent water solubility and were stable inwater over months of storage without aggregation/precipitation.

Stabilities of CN-DOX-IO at Low pH and Against Gastric and SmallIntestine Enzymes

Selectively delivering drugs to the intestine requires conquering thegastric acidic and enzymatic conditions that may prematurely release,degrade and deactivate the drugs The prepared LBL CN-DOX-IO was stableover the pH range of 2.0-8.0. DLS measured hydrodynamic sizes ofCN-DOX-IO (˜25 nm) remained unchanged over this pH range (FIG. 3A)except at the isoelectric point of pH 4.0, in which the hydrodynamicsizes increased to 80 nm. At the isoelectric point of pH 4.0,monodispersed CN-DOX-IO formed reversible clusters, but notprecipitation. This reversible aggregation returned to the singledispersed form (with hydrodynamic size of 25 nm) by adjusting pH tolower/higher than the isoelectric point, due to the presence of abundantpositive/negative charged functional groups in CN. At pH 2.0, which isclose to the pH condition of the stomach fluid, CN-DOX-IO remainedsingle dispersed with a hydrodynamic size of 25 nm for more than 24 h.On the contrary, DOX-IO without the protective casein outer layerprecipitated when pH changed to 2.0 and below, evidenced by the drasticincrease of the hydrodynamic size (FIG. 3B).

To examine the stability of the casein outer layer in CN-DOX-IO againstdigestive enzymes in stomach and its enzymatic-responsive degradation bythe digestive enzymes in the small intestine, CN-DOX-IO was treated withpepsin, which is the protease in the gastric juice that breaks downprotein to peptides, and trypsin, which is a duodenum secreted proteasethat degrades protein or peptides, at different enzyme concentrations atpH 2.0 (for pepsin) and 7.0 (for trypsin). SDS-PAGE gel electrophoresiswas used to examine the protein or peptides digested by the enzymes. Theband of CN in SDS-PAGE gel was still observed (FIG. 3C) after treatmentat pH 2.0 with pepsin (0.1 and 1.5 mg/mL), which represented thephysiological enzyme range of human gastric fluid (0.3-1.3 mgpepsin/mL). DLS measurements of CN-DOX-IO treated with pepsin at pH 2.0also validated the stability of the reported LBL nanostructure in themimicked gastric digestive system. CN-DOX-IO showed persistentdispersibility with unvaried hydrodynamic size after treated with pepsin(FIG. 3E). On the other hand, SDS-PAGE showed different bands whentreating CN-DOX-IO with trypsin at pH 7.0 (FIG. 3D). The amount ofintact CN in DOX-CN-IO decreased while short peptides and fragments ofCN emerged in the SDS-PAGE gel. The size of newly appearing peptidefragments became smaller (lower molecular weight in SDS-PAGE gel) as thetrypsin concentration further increased. DLS measurement showed adecrease in hydrodynamic size of CN-DOX-IO after treated with trypsin(FIG. 3F), which further confirmed the breakdown of the casein outerlayer. These results indicate that LBL CN-DOX-IO nanoparticles canremain intact under the acidic gastric condition with a protectivecasein outer layer resistant to the low pH and pepsin. In addition, thecasein outer layer can be disassembled by the intestine protease trypsinat pH 7.0, thus exposing the inner layer of amphiphilic polymer, whichis loaded with the hydrophobic drug (DOX). Therefore, this LBL deliveryvehicle is suitable for oral drug delivery through the GI tract to theintestine.

Enzymatic Responsive Release of Doxorubicin from CN-DOX-IO

FIG. 4A shows the profiles of DOX released from the CN-DOX-IOnanoparticles and those from DOX-IO at pH 2.0, 5.5 and 7.4 without thepresence of either pepsin or trypsin. Both of the DOX loadednanoparticles exhibited very low release of DOX at pH 5.5 and 7.4,compared to that at pH 2.0. The increased release of DOX at lower pH(2.0) is caused by the protonated DOX, which has a higher solubility inwater. Notably, CN-DOX-IO exhibited a slower and sustained release ofDOX compared with DOX-IO at pH 2.0, referring to the effectiveprotection of DOX by the casein outer layer. When cultured in the mediumsimulating gastric juice (pH 2.0, [Pep]=1.0 mg/mL), the initial rapidrelease of the encapsulated DOX in DOX-IO (62%) was significantlyreduced to about 40% when casein outer layer was applied (FIG. 4B). Theresults implied that the embedded DOX in the inner amphiphilic polymerlayer in DOX-IO was shielded by the pH- and pepsin-stable casein outerlayer. Alternatively, both DOX-IO and CN-DOX-IO showed sustained releaseof DOX in the simulated intestinal juice (pH 7.0, [Try]=2.5 mg/mL). DOXwas released from CN-DOX-IO more effectively (˜30% in 6 h) in the mediumsimulating intestinal juice than from DOX-IO (˜15% in 6 h). The LBLCN-DOX-IO demonstrated a preferential and enzymatic-responsive drugrelease property by preventing the loss of drug in the acidic stomach,thus improving the efficacy of drug delivery to the small intestine.

Uptake of CN-DOX-IO by Caco-2 Cell Monolayer

To investigate the cellular uptake of the released DOX from CN-DOX-IO bythe small intestine in vitro, the monolayer of Caco-2 cells wasincubated with CN-DOX-IO at a dosage of 17.2 μm for different lengths oftime (10, 30 and 60 min). For comparison, cells incubated with DOX-IOand DOX were examined as controls. The intracellular accumulation of DOXwas examined by the fluorescent microscope utilizing the fluorescence ofDOX. After 10 min incubation, fluorescence signal from DOX was observedin the nucleus only in the cells treated with free DOX, owing to thehigh permeability of the small molecules (i.e. DOX), which facilitatedrapid influx into the nucleus. For the cells treated with DOX-IO andCN-DOX-IO, the fluorescence signal of DOX was localized mostly in thecytoplasm instead of the nucleus after 10 min incubation, implying thenanocarriers were uptake through the endocytic process. However, at 30min after incubation, a small amount of DOX was observed to accumulatein the nucleus, which was ascribed to the slow release of DOX fromDOX-IO and CN-DOX-IO. After 60 min incubation, DOX was evidenced in bothcytoplasm and nucleus of the cells treated with DOX-IO and CN-DOX-IO,demonstrating that the sustained release of DOX from the nanoparticlesenabled the continuous accumulation of DOX in the nucleus.

Uptake of CN-DOX-IO by Small Intestine Tissue Samples

To determine the uptake and permeability of CN-DOX-IO in the smallintestine, the jejunum villi of the mouse small intestine was sectionedand incubated with CN-DOX-IO and DOX-IO, respectively, for 1 h. Prussianblue staining for iron revealed the distribution of CN-DOX-IO in thevilli of the small intestine. Intense blue staining was observed in thesacs treated with CN-DOX-IO, in comparison with that of the DOX-IOtreated samples. The results indicate that the casein outer layer mayfacilitate the interaction of nanoparticles with villi to increase thetissue uptake, subsequently enhancing the effective plasmaconcentration.

Distribution and Stability of CN-ICG-IO in Mice Observed with Optical/MRImaging

To study the stability and organ distribution profiles of the developedLBL drug delivery carriers in the GI tract in vivo, noninvasive NIRoptical imaging was used to investigate mice fed with nanoparticles.CN-ICG-IO, in which DOX was substituted with indocyanine green (ICG),was orally administered to mice. ICG-IO was used as the control. Mousestomach and intestine could be identified from the MR images. Theaccumulation of magnetic nanoparticles was confirmed by the change ofMRI contrast, i.e., the almost complete loss of signal or darkening inthe T2-weighted MR images, after oral administration of nanoparticles.At 3 h after oral administration, the NIR signal of ICG was found mostlyin the intestine of the mice administered with CN-ICG-IO. However, thesignal was mainly observed in the area of the stomach for the ICG-IOtreated group, revealing the release of ICG from ICG-IO in the stomach.At 5 h after administration, NIR imaging showed that CN-ICG-IO reachedinto the ileum and spread further in the intestine. However, the NIRsignal in the stomach was still highest in the animals treated withICG-IO, while only a slight increase of NIR signal was observed in thesmall intestine. For the ICG-IO treated group, the signal in the stomachremained even up to 7 h, for the reason that the precipitation of ICG-IOformed and deposited in the crypts of gastric pits. The results fromnoninvasive NIR imaging thus further support that CN-ICG-IO couldsustain in acidic gastric conditions, allowing for preferential deliveryof the payload drugs to the small intestine. In addition, MRI contrastgenerated by iron oxide nanoparticles potentially enables the monitoringof drug delivery by MRI.

1. A method of treating cancer comprising administering an effective amount of a pharmaceutical composition to a subject in need thereof, wherein the pharmaceutical composition comprises a particle; an inner coating on the particle comprising an amphiphilic polymer providing an space for embedding a hydrophobic anti-cancer drug within the inner coating, wherein a hydrophobic drug is in the space of the inner coating; and an outer coating on the inner coating wherein the outer coating is crosslinked casein molecules.
 2. The method of claim 1, wherein the particle is an iron oxide nanoparticle.
 3. The method of claim 1, wherein the amphiphilic polymer comprises maleic acid and octadecene monomers.
 4. The method of claim 1, wherein the anticancer drug is doxorubicin.
 5. The method of claim 4, wherein the crosslinked casein molecules are made by the process of mixing casein and the particle comprising the inner coating in the presences of glutaraldehyde. 